Metab Brain Dis DOI 10.1007/s11011-014-9513-8

RESEARCH ARTICLE

Effects of hyperammonemia on brain energy metabolism: controversial findings in vivo and in vitro Arne Schousboe & Helle S. Waagepetersen & Renata Leke & Lasse K. Bak

Received: 15 January 2014 / Accepted: 14 February 2014 # Springer Science+Business Media New York 2014

Abstract The literature related to the effects of elevated plasma ammonia levels on brain energy metabolism is abundant, but heterogeneous in terms of the conclusions. Thus, some studies claim that ammonia has a direct, inhibitory effect on energy metabolism whereas others find no such correlation. In this review, we discuss both recent and older literature related to this controversial topic. We find that it has been consistently reported that hepatic encephalopathy and concomitant hyperammonemia lead to reduced cerebral oxygen consumption. However, this may not be directly linked to an effect of ammonia but related to the fact that hepatic encephalopathy is always associated with reduced brain activity, a condition clearly characterized by a decreased CMRO2. Whether this may be related to changes in GABAergic function remains to be elucidated. Keywords Hepatic encephalopathy . Brain . Energy metabolism . Ammonia

Introduction Due to the central role of the liver in ammonia metabolism and detoxification as the major organ capable of conversion of ammonia to urea, liver failure is always associated with hyperammonemia, a condition that may result in the clinical A. Schousboe : H. S. Waagepetersen : L. K. Bak (*) Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark e-mail: [email protected] R. Leke Department of Pathology, University of Miami School of Medicine and Veterans Administration Medical Center, Miami, FL 33101, USA

manifestations of hepatic encephalopathy (Butterworth 2002; Lockwood et al. 1979; Ong et al. 2003; Ott et al. 2005). Several studies in the brain in vivo have found this condition to be associated with a decreased oxygen consumption (Alman et al. 1956; Dam et al. 2013; Iversen et al. 2009; Morgan et al. 1980; Philips et al. 1998; Posner and Plum 1960; Strauss et al. 2003) and hence, it is likely that elevated brain concentrations of ammonia may affect energy metabolism (Ott et al. 2005; Rao and Norenberg 2001). Interestingly, it was suggested by Bessman and Bessman (1955) that ammonia-induced withdrawal of α-ketoglutarate from the tricarboxylic acid (TCA) cycle due to glutamate synthesis would decrease the efficacy of the cycle as the energy producing machinery of the mitochondria. Moreover, it was reported that α-ketoglutarate dehydrogenase, a key enzyme of the TCA cycle is inhibited by ammonia (Lai and Cooper 1986; McKhann and Tower 1961) likewise pointing to an inhibitory action of ammonia on energy metabolism. In keeping with this notion it was shown using NMR spectroscopy that brain lactate production was increased during acute liver failure due to inhibition of pyruvate dehydrogenase leading to a reduced oxidative metabolism (Zwingmann et al. 2003). However, other studies of liver failure and cerebral metabolic parameters in rats (Hindfelt et al. 1977; Hindfelt and Siesjo 1971) had shown that ATP levels were only marginally affected and that the TCA cycle was functioning. A possible explanation for this could be a compensatory action of an increased glycolysis, which could maintain the ATP level. Hence, it appears still somewhat contradictory to what extent brain energy metabolism may be affected by hyperammonemia induced by liver failure. In the following section, studies of metabolism of glucose and other substrates performed on cultured neural cells exposed to hyperammonemic conditions will be reviewed. It should be pointed out that most of the experiments reviewed originate from studies performed in acute hyperammonemic conditions

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and that this may be different from results obtained during chronic hyperammonemia.

Effects of ammonia on glucose and lactate metabolism in cultures of astrocytes, and glutamatergic and GABAergic neurons Using neurons and astrocytes cultured from cerebral cortex and cerebellum from mice and carbon-13 labeled glucose or lactate in combination with mass spectrometry and nuclear magnetic resonance spectroscopy it has been studied to what extent cellular metabolism is affected by exposure to an increased concentration of ammonia (Johansen et al. 2007; Leke et al. 2011a). In both neurons and astrocytes [U-13C]glucose will be metabolized in the glycolytic pathway and the TCA cycle giving rise to the labeled keto-acids pyruvate, oxaloacetate and α-ketoglutarate which through aminotransferase reactions will be in equilibrium with the corresponding amino acids alanine, aspartate and glutamate (see, Johansen et al. (2007) for detailed metabolic schemes). The distribution of the number of heavy carbon atoms ([13C]) in these amino acids will provide information about the activities of the glycolytic pathway, TCA cycle as well as the pyruvate carboxylase (PC) reaction which is specifically located in the astrocytes (Shank et al. 1985; Yu et al. 1983). The reason for this is that the isotopomer formation, i.e. M+1, M+2, M+3 and M+4 in aspartate, M+1 to 5 in glutamate and M+3 in alanine will in case of the C-4 and C-5 amino acids reflect the number of turns of the TCA cycle or the activity of PC and in case of alanine monitor glycolytic activity as alanine is likely to be in equilibrium with pyruvate generated by glycolytic cleavage of glucose (for details, see Johansen et al. (2007)). Exposure of cerebellar neuronal cultures consisting mainly of glutamatergic neurons (Drejer and Schousboe 1989; Sunol et al. 2010) to ammonia led to a significant increase in the amounts of the three amino acids in question and to an increase in carbon-13 labeling of the amino acids. In case of glutamate the increase in labeling was most pronounced for the M+5 isotopomer which is produced after several turns of the TCA cycle. It can therefore be concluded that the activity of the TCA cycle is enhanced by exposure of the neurons to ammonia, which is in contrast to previous indications that ammonia inhibits important enzymes of this metabolic cycle (see above). The increased labeling in alanine points to an enhanced glycolysis, which may be in keeping with the observation in vivo, that lactate production is enhanced by hyperammonemia caused by acute liver failure (Zwingmann et al. 2003). In astrocytes cultured from cerebellum ammonia was found to have effects similar to those seen in the neurons indicating that also astrocytic glycolysis and TCA cycle activity were enhanced by hyperammonemia. Interestingly, in the astrocytes the production of uniformly labeled glutamate

produced by the PC metabolic pathway was increased by ammonia. This may reflect the need for de novo synthesis of α-ketoglutarate which requires pyruvate carboxylation (see, Schousboe et al. 2013). This effect of ammonia on the activity of PC has also been observed in a more recent study particularly designed to study ammonia-induced production of glutamine (Dadsetan et al. 2011). In cultures of cerebral cortical neurons and in co-cultures of these neurons and astrocytes representing the GABAergic neurotransmitter system (Drejer et al. 1987; Leke et al. 2008) it was found that exposure to ammonia enhanced neuronal TCA cycle metabolism and switched the astrocytic TCA cycle from mainly producing energy to producing αketoglutarate involving the anaplerotic pyruvate carboxylation to allow a net synthesis of glutamine aimed at ammonia detoxification (Leke et al. 2011a). Interestingly, it was additionally observed that increased glycolytic activity was utilized to allow a net production of alanine. In analogy to the results obtained using the glutamatergic neurons in culture, no indication was found of an inhibitory effect hyperammonemia on energy metabolism. Interestingly, however, the ATP content of the cells both in mono- and co-cultures of the neurons could be maintained by glucose in the presence of ammonia while replacing glucose with either lactate or βhydroxybutyrate led to a reduction of the ATP level after exposure to ammonia. This indicates that ammonia does have an effect demanding an increased availability of ATP that can be fulfilled by the combined activity of glycolysis and the TCA cycle but not the TCA cycle alone. An enhanced glycolytic activity induced by ammonia is supported by the finding that the lactate production was increased under these conditions (Leke et al. 2011a), a finding compatible with observations in vivo (Zwingmann et al. 2003). The increase in glycolytic activity may be a compensatory mechanism ameliorating the inhibitory action of ammonia on the malate-aspartate shuttle reported previously (Hertz and Kala 2007; Hindfelt et al. 1977). It may be noted that in a study of the effects of ammonia in cultures of cerebral cortical neurons conducted by Kala and Hertz (2005) no increase in lactate production was observed whereas in astrocyte cultures exposure to ammonia did lead to an increased production of lactate. Actually, in the study by Leke et al. (2011a), the ammonia-induced increase in lactate production was much more pronounced in the cocultures compared to that in the neuronal cultures indicating that the astrocytes are responsible for the major part of the ammonia-induced increase in glycolytic activity. In agreement with this, a study by Hetz et al. (1987) of the effect of ammonia on cerebral cortical astrocytes showed a small albeit not significant increase in glucose utilization. Interestingly, however, pyruvate oxidation was decreased in the absence of glutamine in the medium while it was unaffected when glutamine was present, a finding possibly related to the regulatory mechanisms related to the demand of anaplerosis via

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pyruvate carboxylation for generation of α-ketoglutarate as discussed above. It may also be related to the fact that glutamate is a major metabolic fuel in astrocytes (Schousboe et al. 2013) via the action of glutamate dehydrogenase. Interestingly this oxidation of glutamate has been shown to be reduced by a high concentration of ammonia (Yu et al. 1984).

Hyperammonemia and GABAergic neurotransmission It is generally accepted that hyperammonemia and HE affect multiple neurotransmitter systems including the GABAergic system (Albrecht and Jones 1999; Butterworth 2000; Jones 2003; Jones and Basile 1998; Jones et al. 1984). Regarding the GABAergic neurotransmission, it has been shown that GABA-receptors, -release, -uptake, and -metabolism may be affected (Basile et al. 1988; Bender and Norenberg 2000; Cauli et al. 2007; Li et al. 2005; Oja et al. 1993; Wysmyk et al. 1992). A recent study of the effect of an HE-like condition in rats induced by bile duct ligation has, however, shown that the expression of mRNA for the two glutamic acid decarboxylase (GAD) isozymes responsible for GABA biosynthesis was not affected by this condition (Leke et al. 2013). Nevertheless, it should be noted that previous studies of GABA synthesis in different rat models of HE have indicated a reduced activity of GAD associated with this condition (Wysmyk-Cybula et al. 1986; Zeneroli et al. 1982). Using the co-culture system of GABAergic neurons and astrocytes developed by Leke et al. (2008) and the astrocyte specific substrate [13C]acetate (see, Waniewski and Martin 1998), Leke et al. (2011b) have studied in detail biosynthesis of transmitter GABA derived from astrocytically derived glutamine. Since acetate is only metabolized in the astrocytic compartment in this culture system and glutamine can only be synthesized in the astrocytes due to the cell-specific localization of glutamine synthethase (Norenberg and MartinezHernandez 1979), any [13C]-labeling in GABA can only occur as a result of transfer of labeled glutamine from the astrocytes to the neurons and subsequent synthesis of GABA from this pool of glutamine (for details, see Leke et al. 2011b). Additionally, experiments were performed in which the cultures were incubated in [U-13C]glutamine. It should be noted that it has previously been demonstrated that conversion of glutamine to GABA via the concerted action of phosphate activated glutaminase and GAD (see, Leke et al. 2011b) to a considerable extent (60 %) involves the TCA cycle and this pathway is referred to as the indirect pathway in contrast to the direct pathway not involving participation of the TCA cycle (for details, see Schousboe et al. 2013). The indirect pathway represent s synthesis of n eu ro t ran s mi t t er GABA (Waagepetersen et al. 2001). Interestingly, it was found that the hyperammonemic condition had little if any effect on the biosynthetic pathway for GABA. However, in bile duct

ligated rats where GABA synthesis was monitored using labeled acetate, it was found that this condition preferentially increased GABA synthesis via the indirect pathway which would indicate that hyperammonemia could increase the availability of neurotransmitter GABA (Leke et al 2011b). This may be in contrast to the observation that in a synaptosomal preparation no effect of ammonia was seen on GABA release (Erecinska et al. 1987). This may not, however, be completely comparable to the in vivo situation. A possible enhanced GABAergic activity may be interesting in the light of the fact that HE and hyperammonemia are associated with reduced brain activity leading to cognitive impairment (Cauli et al. 2009; Leke et al. 2012). It should be mentioned that the effects of ammonia on cognition may only be observed during chronic conditions of hyperammonemia.

Concluding remarks The discussions presented above may not solve the discrepancies between the studies indicating an inhibitory effect of ammonia on α-ketoglutarate dehydrogenase and the findings in cultured neural cells as well as in the brain in vivo that ammonia does not seem to inhibit the TCA cycle activity as also discussed by Ott and Vilstrup (2014) in this issue of Metabolic Brain Disease (in addition, these authors offers an elaborate discussion of the current hypotheses on how liver disease affects cerebral function). Taking this into consideration it may seem peculiar that it has been consistently reported that HE and hyperammonemia lead to reduced cerebral oxygen consumption, i.e. reduced CMRO2. This apparent enigma may be solved by pointing out that HE is always associated with reduced brain activity and even coma, a condition clearly characterized by a decreased CMRO2 (Gjedde et al. 2002). Whether or not this may be related to the changes in GABAergic function remains to be elucidated but it appears possible that a direct inhibitory action of ammonia on mitochondrial activity and energy production may not be a requirement to explain the reduced CMRO2 associated with HE and hyperammonemia.

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